This invention relates to novel microorganism, a biologically pure culture of strain UK 788 of Bacillus stearothermophilus, and a process for producing a useful enzyme selected from the group consisting of a heat-resistant polynucleotide phosphorylase, heat-resistant maleate dehydrogenase, heat-resistant glucokinase, heat-resistant glucose-6-phosphate dehydrogenase, and heat-resistant pyruvate kinase.
Since bacterial cells are smaller in size than yeast or fungal cells, they are difficult to collect from a culture, so they are usually collected by centrifugation rather than by filtration. But the rate of sedimentation of cells in centrifugation is proportional to the square of the cell diameter (see Sekiyu Hakko, Petroleum Fermentation, p. 102, 1970, Saiwai Publishing Company), so the recovery of bacterial cells from the fermentation broth is more expensive than recovery of yeast and fungal cells and is very disadvantageous in an industrial operation. According to the estimate by Daniel I. C. Wang, the cost of recovery of bacteria is about 3.8 times the cost of recovery of yeast (see Chemical Engineering, Vol. 15, p. 99, 1968). Therefore, several methods have been proposed for recovery of bacterial cells and among them is flocculation of cells with a flocculant such as ferric chloride, calcium chloride or polymeric flocculant, or modifying the cell protein to an easily collectable form by heating or treatment with a strong acid or base. These techniques are effective when the cell is not the end product, but when the cell per se or the components in the cell are the end product, they are not effective because the flocculant contaminates the product, or the components in the cell are denatured.
Polynucleotide phosphorylase, one example of the endoenzymes, is used for synthesis of ribo-homopolymers or copolymers of nucleotide, a variety of analog polynucleotides, oligonucleotides having a fixed base arrangement, and for decomposition of RNA. The enzyme also finds much use in the study of the structure and physical properties of nucleic acids. Polynucleotide phosphorylase is conventionally obtained as an extract from Micrococcus luteus (see Procedures in Nucleic Acid Research, Vol. 2, p. 896, 1971), but the polynucleotide phosphorylase isolated from that microorganism is so labile that it is unsuitable for use even in laboratory-scale research, to say nothing of use on an industrial scale.
Seikagaku (Biochemistry), Vol. 47, p. 738 (1975) describes a method of producing a highly heat-stable, heat-resistant polynucleotide phosphorylase from a thermophilic bacterium Thermus thermophilus, and Nucleic Acids Research, Vol. 3, p. 219 (1976) describes a method of producing the same enzyme from a thermophilic bacterium Bacillus stearothermophilus.
Maleate dehydrogenase which produces oxaloacetic acid from malic acid using nicotinamide adenine nucleotide as a coenzyme has recently been much in demand as an enzyme for use in clinical diagnosis. This enzyme is usually obtained as a purified extract from animal tissues, say, bovine heart (see Methods in Enzymology, Vol. 13, p. 99), but maleate dehydrogenase isolated from animal tissues is labile and is not easy to handle. Therefore, a supply of a stable maleate dehydrogenase was desired, and the Journal of Biological Chemistry, Vol. 242, p. 1548, 1967 and Biochemical Journal, Vol. 177, p. 441, 1979 have described a method of producing a highly heat-stable, heat-resistant maleate dehydrogenase from a thermophilic bacterium Bacillus stearothermophilus.
Glucokinase and glucose-6-phosphate dehydrogenase are used to measure the glucose or hexokinase level in the body fluid, which is an important factor in clinical analysis. They are also used to determine the glucose or fructose level in foods which is also an important parameter to know in the manufacture of invert sugar. However, known glucokinase and glucose-6-phosphate dehydrogenase are labile, too (see Methods in Enzymology, Vol. 42, pp. 6-39, 1975 ed. by W. A. Wood, and Advances in Enzymology, Vol. 48, pp. 97-191, 1979, ed. by Alton Meister). The only known exception is the glucokinase described in FEBS Letters, Vol. 37, pp. 212-216, 1973; obtained from a thermophilic Bacillus stearothermophilus, this enzyme is heat stable and has long storage stability.
Pyruvate kinase is found in all living tissues such as animals, plants, yeast and bacteria that metabolize carbohydrates by glycolysis or fermentation. In the living cells, the enzyme catalyzes the reaction of forming adenosine triphosphate and pyruvic acid from phosphoenol-pyruvic acid (PEP) and adenosine diphosphate (ADP), so it is used for determination of PEP and ADP. The conventional crystal of pyruvate kinase is available as an isolate from rabbit muscle or swine heart (see Methods in Enzymology, Vol. 1, p. 435, and Journal of Biological Chemistry, Vol. 234, p. 2428, 1955), but such pyruvate kinase is so labile that it is not suitable for industrial use. Japanese Patent Application (OPI) NO. 9392/78 (the term "OPI" as used herein refers to a "published unexamined Japanese patent application") describes a method of producing a highly heat-stable, heat-resistant pyruvate kinase from a thermophilic Thermus thermophilus, and Seikagaku (Biochemistry), Vol. 44, p. 649, 1972, describes a method of producing the same enzyme from a thermophilic Bacillus stearothermophilus.
To obtain these useful endoenzymes, cultured cells must be first collected by centrifugation or other suitable means before the desired enzyme is extracted from the cells by ultrasonic treatment or physical breaking.
The collection of cultured cells is difficult, as was already mentioned, because of the low specific gravity of bacteria. In addition, bacteria belonging to the genus Bacillus have a relatively hard membrane which can be broken so slightly that the efficiency in extraction of a desired enzyme is low. For these reasons, it has been difficult to produce these enzymes on an industrial scale.